Expression of alkaline phosphatase by a B-cell hybridoma and its modulation during cell growth and apoptosis

The 7TD1 B-cell hybridoma was found to spontaneously express Alkaline Phosphatase (ALP), an Enzyme which is produced by splenic B lymphocytes once optimally activated. Determination of ALP levels during cell growth and departure to Apoptosis showed fluctuations. Following a temporary increase within the first 24 h, Enzyme expression was maintained at high levels during the early proliferation stage, and then declined from 3 to 4 days in mid-exponential phase to basal levels at day 6 when living cells were no longer detectable and the apoptotic process was completed. The protein synthesis inhibitor, cycloheximide (1 microg/ml), decreased ALP production while stimulating a strong Apoptosis of 7TD1 cells, within 4 h. Aphidicolin (1 microg/ml) maintained ALP production and provoked a release of ALP activity into the surrounding medium; it also induced Apoptosis, but with a 24 h delay. Quantification of Apoptosis and ALP expression by flow cytometry, after simultaneous staining of DNA with Hoechst 33342 and ALP with naphthol AS-TR phosphate/Fast Red RC fluorescent reagent, revealed cell cycle modulation of ALP expression, its activity increasing as 7TD1 cells progressed from G1 phase into S and G2/M phases of the cell cycle in control as well as in drug-treated cells. Kinetics of drug-induced Apoptosis and higher expression of ALP associated preferentially with active cell growth during the prevention stage of Apoptosis suggested a possible link between cellular ALP expression and cell survival.